Development of a highly sensitive quantification system for assessing dna degradation and quality in forensic samples

ABSTRACT

A process of quantifying the extent of degradation present in a human DNA sample is described. The process makes use of a real time PCR system to separately quantitate within a sample a first retrotransposon interspersed element and a relatively longer second retrotransposon interspersed element, where the longer element is expected to be disrupted at a faster pace than is the shorter element as the sample degrades. In one embodiment, the process makes use of the appearance of the relatively young (on an evolutionary scale) Alu Yb-lineage subfamily sequences appearing in every human genome and their virtual absence in non-human samples. In a preferred embodiment, the process quantifies longer 290 bp sequences of “SVA” elements and shorter 80 bp sequences of Alu Yb8-lineage. Newly designed primers and TaqMan probes that are useful in the process are presented. A related process additionally quantifies male specific human DNA.

CLAIM OF PRIORITY

This application makes reference to, incorporates the same herein, andclaims all benefits accruing under 35 U.S.C. §119(e) from an applicationfor DEVELOPMENT OF A HIGHLY SENSITIVE QUANTIFICATION SYSTEM FORASSESSING DNA DEGRADATION AND QUALITY IN FORENSIC SAMPLES, earlier filedin the United States Patent and Trademark Office as a provisionalapplication under 35 U.S.C. §111(b) on Aug. 13, 2012, and duly assignedSer. No. 61/682,507, another application of the same title earlier filedin the United States Patent and Trademark Office as a provisionalapplication under 35 U.S.C. §111(b) on Feb. 21, 2013, and duly assignedSer. No. 61/767,668 and a third application of the same title earlierfiled in the United States Patent and Trademark Office as a provisionalapplication under 35 U.S.C. §111(b) on Mar. 15, 2013, and duly assignedSer. No. 61/793,595.

BACKGROUND OF THE INVENTION

1. Field of the Invention

A process for determining the extent of environmental degradation of ahuman DNA sample by using newly identified target elements in a realtime polymerase chain reaction system is disclosed.

2. Description of the Related Art

In the last recent years, real-time polymerase chain reaction (PCR)chemistry has become the standard for reliably quantifying the amount ofgenomic and amplifiable DNA in a forensic sample. Commonly used systemsinclude the assessment of total human and male DNA. Examples areQuantifiler® from Life Technologies Corporation, Plexor® from PromegaCorporation and Quantiplex® from Qiagen. Currently there are severaldifferent approaches used for fluorescence-based quantification assays,including SYBR® Green, Plexor®, TaqMan®, AmpliFluor®, Quantifiler® andQuantiplex®.

Interest has recently grown in using real-time PCR methods to evaluatethe extent of degradation of a DNA sample. This may be done using twonuclear DNA targets: a short multi-copy sequence and a long multi-copysequence. Because the long target sequence will degrade more rapidlythan will the short target sequence as a sample is compromised, theratio of the quantity of the short target to the long target willprovide an assessment of the extent of degradation in the sample.Studies on the assessment of degraded DNA in a forensic sample have beenpublished using Alu or mini-satellite targets. However, the assays ofprevious studies either lack in sensitivity or do not exhibit high PCRefficiencies. Forensic samples vary widely in quantity and quality,making the goal of developing and validating a real-time PCR system forthe purposes of quantitating the DNA in these samples and determiningthe extent of their degradation a challenging one.

The recent advances in mini short tandem repeat (STR) analysis systemshave now made it possible to analyze highly compromised samples.Investigators have made great strides in the development of STRamplicons that, compared with traditional STR amplicons, are reduced insize and can be used effectively on DNA samples that have beensignificantly degraded (see, e.g., J. M. Butler, et al., J. ForensicSci. 48(5): 1054-1064 (2003); T. J. Parsons, et al., Forensic ScienceInternational: Genetics 1: 175-179 (2007)).

Alu are Short Interspersed Elements (SINE), approximately 300 bpinsertions which are distributed throughout the human genome in largecopy number. The evolution of Alu elements in the human genome over timehas made Alu elements well suited for the task of distinguishing humanDNA from non-human DNA and for doing testing that is desired to bespecific to human DNA. A recent study reports an evaluation of thequality assessment of degraded DNA samples using a Ya5-lineage Alugenetic element (J. A. Nicklas, et al., J. Forensic Sci. 57(2): 466-471(2012)). A multi-copy intra-Alu based approach for quantifying humanspecific DNA in an evidence sample has been successfully used to obtainDNA quantification with high sensitivity (J. A. Walker, et al., Anal.Biochem. 337: 89-97 (2005)).

The average age of Yb-lineage subfamily elements is estimated as 2.39million years. It is estimated that the human genome contains over 1800Alu Yb family elements and, out of those, approximately 50% are from theYb8 subfamily. The Alu Yb8 system is known for the presence of a largenumber of fixed insertions. It has been reported that only 20% of theYb-lineage Alu elements are polymorphic for insertion presence orabsence in the human genome (A. B. Carter, et al., Human Genomics 1(3):1-13 (2004)). Because a large number of these fixed elements are presentin every human genome, the individual specific variation possible whenusing a multi-copy target quantification system is minimized.

In 1994, Shen, et al., identified a new composite retroposon when theystudied the structure of the RP gene (Shen, et al., J. Biol. Chem.269(11): 8466-8476 (1994)). This new retroposon consisted of the SINE-Relement together with a stretch of sequence that shares sequencesimilarity with Alu sequences. Thus, it was named “SVA” after its maincomponents, Short Interspersed Elements (SINE), Variable Number TandemRepeats (VNTR) and Alu. SVA elements contain the hallmarks ofretrotransposons, in that they are flanked by target site duplications(TSDs), terminate in a poly(A) tail and are occasionally truncated andinverted during their integration into the genome.

SUMMARY OF THE INVENTION

One object of the present invention is to provide a method ofquantifying the extent of degradation present in a human DNA sample.

Another object of the invention is to provide a method for quantitatingthe total amount of human DNA as well as the male DNA in a sample.

Another object of the invention is to provide an internal positivecontrol that will offer increased confidence in the results of the DNAdegradation determination by providing an additional assessment for thepresence of PCR inhibitors in the sample.

Another object of the invention is to provide a convenient means foranalysts to choose from among multiple DNA samples the best one forfurther analytical attention.

Another object of the invention is to provide an improved means forselecting the optimum analytical method to employ on a particular DNAsample, based on the extent of its degradation.

Another object of the invention is to provide a means of assessing theextent of admixture of non-human DNA with the human DNA sample beingtested.

Another object of the invention is to provide a means of assessing theextent of admixture of male and female DNA in the sample being tested.

These and other objects may be attained, in one embodiment of thepresent invention, from a process for quantitating a human DNA in asample in order to assess the extent of degradation of the DNA thereinby providing a sample to be analyzed, using a real time polymerase chainreaction system to separately quantitate within the sample a firstretrotransposon interspersed element and a second retrotransposoninterspersed element, the first retrotransposon interspersed elementbeing an Alu element and the second retrotransposon interspersed elementbeing an SVA element of the RP gene, and calculating a ratio of anoccurrence within the sample of the first retrotransposon interspersedelement to an occurrence of the second retrotransposon interspersedelement.

In certain embodiments, the quantitation of the first retrotransposoninterspersed element and the second retrotransposon interspersed elementmay be performed simultaneously.

In certain embodiments, the ratio of an occurrence within the sample ofthe first retrotransposon interspersed element to an occurrence of thesecond retrotransposon interspersed element may be used to determine anextent of degradation of the DNA in the sample.

In certain embodiments, the second retrotransposon interspersed elementmay comprise at least three times as many base pairs as are comprised bythe first retrotransposon interspersed element.

In certain embodiments, the process of the invention may furthercomprise the steps of providing a first probe comprising a first moietycapable of fluorescence at a first diagnostic wavelength and a firstquencher capable of quenching the first moiety fluorescence, the firstprobe being targeted to a first retrotransposon interspersed element,providing a second probe comprising a second moiety capable offluorescence at a second diagnostic wavelength and a second quenchercapable of quenching the second moiety fluorescence, the second probebeing targeted to a second retrotransposon interspersed element,providing at least one primer that is useful in the real-time polymerasechain reaction system, the system being capable of amplification of aDNA sample, providing a Taq polymerase enzyme capable of catalyzing theformation of a nucleic acid sequence that is complimentary to onepresent in the sample, the polymerase enzyme being capable both ofcleaving the first probe to separate the first fluorescent moiety fromthe first quencher and of cleaving the second probe to separate thesecond fluorescent moiety from the second quencher, treating the samplewith the first probe and the second probe, amplifying the sample usingthe at least one primer and the Taq polymerase enzyme by means of thereal-time polymerase chain reaction system, the real time polymerasechain reaction system including a plurality of polymerase chain reactioncycles, illuminating the sample during each real time polymerase chainreaction cycle using an excitation source capable of inducingfluorescence in both the first moiety and the second moiety, measuringthe fluorescence emitted from the first moiety and the fluorescenceemitted from the second moiety for each real time polymerase chainreaction cycle, determining a threshold cycle number for the firstretrotransposon interspersed element and the second retrotransposoninterspersed element, and comparing the determined threshold cyclenumbers with standard curves for each of the first retrotransposoninterspersed element and the second retrotransposon interspersed elementto determine a concentration for each of the first retrotransposoninterspersed element and the second retrotransposon interspersed elementwithin the sample. Because the longer retrotransposon interspersedelement sequence is degraded more rapidly in the environment than is theshorter one, this ratio is diagnostic of the extent of DNA degradation.

In certain embodiments, the process for quantitating human DNA in asample may further comprise providing at least one primer selected fromthe group consisting of a forward primer labeled SEQ ID NO: 5 and areverse primer labeled SEQ ID NO: 6 (primers for the 79 base pair Yb8Alu fragment) and providing at least one primer selected from the groupconsisting of a forward primer labeled SEQ ID NO: 8, a forward primerlabeled SEQ ID NO: 11, a forward primer labeled SEQ ID NO: 14, a reverseprimer labeled SEQ ID NO: 9, a reverse primer labeled SEQ ID NO: 12, areverse primer labeled SEQ ID NO: 13 and a reverse primer labeled SEQ IDNO: 15 (primers for the 290 base pair SVA fragment):

(SEQ ID NO: 5) 5′ GGAAGCGGAGCTTGCAGTGA 3′ (SEQ ID NO: 6) 5′AGACGGAGTCTCGCTCTGTCGC 3′ (SEQ ID NO: 8) 5′ TGGGATCCTGTTGATCTGTGACCT 3′(SEQ ID NO: 9) 5′ GATTTGGCAGGGTCATGGGACAAT 3′ (SEQ ID NO: 11) 5′ATGTGCTGTGTCCACTCAGGGTTA 3′ (SEQ ID NO: 12) 5′TTCTTGGGTGTTTCTCACAGAGGG 3′ (SEQ ID NO: 13) 5′ATTCTTGGGTGTTTCTCACAGAGG 3′ (SEQ ID NO: 14) 5′ CCAACCCTGTGCTCTCTGAAAC 3′(SEQ ID NO: 15) 5′ TTTGGCAGGGTCATGGGACAA 3′.

Alternatively, in another embodiment, the approximately 80 base pair Yb8Alu fragment may be paired with the approximately 250 base pair Alu Ya5fragment as target elements in the inventive process. In theseembodiments, the process for quantitating human DNA in a sample mayfurther comprise providing at least one primer selected from the groupconsisting of a forward primer labeled SEQ ID NO: 5 and a reverse primerlabeled SEQ ID NO: 6 (primers for the 79 base pair Yb8 Alu fragment) andproviding at least one primer selected from the group consisting of aforward primer labeled SEQ ID NO: 1, a forward primer labeled SEQ ID NO:2 and a reverse primer labeled SEQ ID NO: 3 (primers for the 250 basepair Alu Ya5 fragment):

(SEQ ID NO: 5) 5′ GGAAGCGGAGCTTGCAGTGA 3′ (SEQ ID NO: 6) 5′AGACGGAGTCTCGCTCTGTCGC 3′ (SEQ ID NO: 1) 5′ TCACGCCTGTAATCCCAGCACTT 3′(SEQ ID NO: 2) 5′ ACGCCTGTAATCCCAGCACTTTG 3′ (SEQ ID NO: 3) 5′TCTGTCGCCCAGGCTGGAGT 3′.

In certain embodiments, the first probe may have a sequence labeled SEQID NO: 7 (for the 79 base pair Yb8 Alu fragment), and the second probemay be selected from the group consisting of a sequence labeled SEQ IDNO: 4 (for the 250 base pair Alu Ya5 fragment) and a sequence labeledSEQ ID NO: 10 (for the 290 base pair SVA fragment):

(SEQ ID NO: 4) 5′ ATCACGAGGTCAGGAGATCGAGACCAT 3′ (SEQ ID NO: 7) 5′AGATTGCGCCACTGCAGTCCGCAG 3′ (SEQ ID NO: 10) 5′AAGGGCGGTGCAAGATGTGCTTTGTT 3′.

In certain embodiments, the real-time polymerase chain reaction systemmay operate under the following approximate conditions: 95° C. for 10minutes; 32-40 cycles of: 95° C. for 15 seconds, 61° C. for 70-120seconds.

In certain embodiments, an internal positive control may be added to thesample to form a sample mixture. A real-time polymerase chain reactionsystem may then be used to quantitate the internal positive controlwithin the sample mixture, and an occurrence within the sample mixtureof the second retrotransposon interspersed element may be compared withan occurrence within the sample mixture of the internal positivecontrol. The latter comparison may then be used to determine an extentto which the process was affected by the presence of an inhibitor in thesample.

In certain embodiments, the quantitation of the first retrotransposoninterspersed element, the second retrotransposon interspersed elementand the internal positive control may be performed simultaneously.

In some embodiments, the internal positive control may comprise asynthetic nucleotide sequence.

In certain embodiments, the process for quantitating human DNA in asample may comprise providing a primer for the internal positivecontrol, the primer for the internal positive control being selectedfrom the group consisting of a sequence labeled SEQ ID NO: 16, asequence labeled SEQ ID NO: 17, a sequence labeled SEQ ID NO: 18 and asequence labeled SEQ ID NO: 19:

(SEQ ID NO: 16) 5′ AAAGATCCTGCCAACAGGACAGTG 3′ (SEQ ID NO: 17) 5′ACAGACGGTATAGAGACCAATCAG 3′ (SEQ ID NO: 18) 5′ GCATAAAGATCCTGCCAACAG 3′(SEQ ID NO: 19) 5′ ACCAAAGTGCTGCAGAAATAC 3′.

In certain embodiments, the process for quantitating human DNA in asample may comprise providing a third probe, the third probe comprisinga third moiety capable of fluorescence at a third diagnostic wavelengthand a third quencher capable of quenching the third moiety fluorescence,the third probe being targeted to the positive internal control, the Taqpolymerase enzyme being further capable of cleaving the third probe toseparate the third fluorescent moiety from the third quencher, the thirdprobe having a sequence labeled SEQ ID NO: 20:

(SEQ ID NO: 20) 5′ AGGCAGAGATTGCACTGCCTTAAAGTGG 3′.

In certain embodiments, the first retrotransposon interspersed elementand the second retrotransposon interspersed element may independently beone of a SINE target sequence, a LINE (long interspersed elements)target sequence and a SVA target sequence.

In certain embodiments, the first retrotransposon interspersed elementmay consist of 40 to 150 base pairs, and the second retrotransposoninterspersed element may consist of 120 to 400 base pairs.

In certain embodiments, the sensitivity of the DNA quantitation may bein the range of from about 5 pg to about 9 pg.

In certain embodiments, the efficiency of the real time polymerase chainreaction system with respect to each of the first retrotransposoninterspersed element and the second retrotransposon interspersed elementmay be at least about 80%.

In certain embodiments, the process step of using a real time polymerasechain reaction system may include the preparation of standard curves forthe quantitation of the first retrotransposon interspersed element andthe quantitation of the second retrotransposon interspersed element. Thestandard curves may each be a plot of a threshold cycle vs. a quantityof DNA, and each may have an R² value of at least 0.99.

In preferred embodiments, the real time polymerase chain reaction systemthat is used in the inventive process for quantitating human DNA in asample may be substantially unreactive to non-primate DNA in the sample.

In certain embodiments, the DNA in a tested sample may have beendegraded by one of mechanical means, chemical means and environmentalmeans.

In embodiments of the invention, the real time polymerase chain reactionsystem may be one of SYBR® Green, TaqMan® and AmpliFluor®.

In certain other embodiments, a process for quantitating total human DNAand male specific DNA in a sample in order to assess the extent ofdegradation of the DNA therein according to the present invention maycomprise the steps of providing a sample to be analyzed, using a realtime polymerase chain reaction system to separately quantitate withinthe sample a male specific DNA sequence, a first retrotransposoninterspersed element and a second retrotransposon interspersed element,the male specific DNA sequence being a 90 bp Y-chromosome specific DNAsequence, the first retrotransposon interspersed element being an Aluelement, the second retrotransposon interspersed element being an SVAelement of the RP gene, and calculating a ratio of an occurrence withinthe sample of the first retrotransposon interspersed element to anoccurrence of the second retrotransposon interspersed element.

In certain embodiments of the process for quantitating total human DNAand male DNA in a sample, the first retrotransposon interspersed elementmay be one of a SINE target sequence, a LINE target sequence and a SVAtarget sequence consisting of 40 to 150 base pairs, the secondretrotransposon interspersed element may be one of a SINE targetsequence, a LINE target sequence and a SVA target sequence consisting of120 to 400 base pairs, and the male target may be a region of a human Ychromosome DNA containing a 90 base pair sequence which is deleted on ahuman X-chromosome in an X-Y chromosome homologous region.

In certain embodiments of the process for quantitating total human DNAand male specific DNA in a sample, the first retrotransposoninterspersed element may be a target sequence that has about 80 basepairs and is an Alu element of subfamily Yb8, the second retrotransposoninterspersed element may be a target sequence that has about 290 basepairs and is an SVA element of the RP gene, and the male specific DNAsequence may be a region of a human Y chromosome DNA containing a 90base pair sequence which is deleted on a human X-chromosome in an X-Ychromosome homologous region.

In certain embodiments, the process for quantitating total human DNA andmale specific DNA in a sample may further comprise providing at leastone primer selected from the group consisting of a forward primerlabeled SEQ ID NO: 5 and a reverse primer labeled SEQ ID NO: 6 (primersfor the Yb8 Alu fragment), providing at least one primer selected fromthe group consisting of a forward primer labeled SEQ ID NO: 8, a forwardprimer labeled SEQ ID NO: 11, a forward primer labeled SEQ ID NO: 14, areverse primer labeled SEQ ID NO: 9, a reverse primer labeled SEQ ID NO:12, a reverse primer labeled SEQ ID NO: 13 and a reverse primer labeledSEQ ID NO: 15 (primers for the SVA sequence):

(SEQ ID NO: 5) 5′ GGAAGCGGAGCTTGCAGTGA 3′ (SEQ ID NO: 6) 5′AGACGGAGTCTCGCTCTGTCGC 3′ (SEQ ID NO: 8) 5′ TGGGATCCTGTTGATCTGTGACCT 3′(SEQ ID NO: 9) 5′ GATTTGGCAGGGTCATGGGACAAT 3′ (SEQ ID NO: 11) 5′ATGTGCTGTGTCCACTCAGGGTTA 3′ (SEQ ID NO: 12) 5′TTCTTGGGTGTTTCTCACAGAGGG 3′ (SEQ ID NO: 13) 5′ATTCTTGGGTGTTTCTCACAGAGG 3′ (SEQ ID NO: 14) 5′ CCAACCCTGTGCTCTCTGAAAC 3′(SEQ ID NO: 15) 5′ TTTGGCAGGGTCATGGGACAA 3′,and providing at least one primer selected from the group consisting ofa forward primer labeled SEQ ID NO: 25 and a reverse primer labeled SEQID NO: 26:

(SEQ ID NO: 25) 5′ CAATGTG[CTAGGCTCTAGGAATAC 3′ (SEQ ID NO: 26) 5′AAGAGTGTCATGGCTCAAAGAG 3′.

In certain embodiments, the process for quantitating total human DNA andmale specific DNA in a sample may further comprise providing a probe forthe male specific DNA target sequence, the probe having a sequencelabeled SEQ ID NO: 27:

(SEQ ID NO: 27) 5′ AGAGAGTATGACAAACATGGCATGGGC 3′.

In certain embodiments, the process for quantitating total human DNA andmale specific DNA in a sample may further comprise adding an internalpositive control to the sample to form a sample mixture, using the realtime polymerase chain reaction system to quantitate the internalpositive control within the sample mixture, comparing an occurrencewithin the sample mixture of the second retrotransposon interspersedelement with an occurrence within the sample mixture of the internalpositive control, and using the comparison to determine an extent towhich the process was affected by the presence of an inhibitor in thesample.

In certain embodiments, the process for quantitating total human DNA andmale specific DNA in a sample may further comprise providing at leastone primer selected from the group consisting of a forward primerlabeled SEQ ID NO: 21, a reverse primer labeled SEQ ID NO: 22, and areverse primer labeled SEQ ID NO: 23 (primers for a specificallytailored internal positive control):

(SEQ ID NO: 21) 5′ GCATAAAGATCCTGCCAACAG 3′ (SEQ ID NO: 22) 5′GCCCGAACTTCCAACACTAT 3′ (SEQ ID NO: 23) 5′ ATTGTTCCTCCTGCCTGATT 3′.

In certain embodiments, the process for quantitating total human DNA andmale specific DNA in a sample may further comprise providing a probe forthe positive internal control, the probe having a sequence labeled SEQID NO: 24:

(SEQ ID NO: 24) 5′ ACAGTGTCAGGCAGAGATTGCACT 3′.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor.

Copies of this patent or patent application publication with colordrawing(s) will be provided by the Office upon request and payment ofthe necessary fee.

A more complete appreciation of the invention, and many of the attendantadvantages thereof, will be readily apparent as the same becomes betterunderstood by reference to the following detailed description whenconsidered in conjunction with the accompanying figures, wherein:

FIG. 1 is a flowchart illustrating a process for determining the extentof environmental degradation of a human DNA sample according to thepresent invention.

FIGS. 2A and 2B show melt curve analyses for the Yb8 and SVA target,respectively.

FIG. 3 shows observed threshold cycle values vs. expected concentrationsof standards for the gDNA sensitivity standard dilutions.

FIG. 4 shows fold changes between each replicate of the standarddilutions, based on at least 34 readings at each DNA concentration.

FIGS. 5A and 5B show observed threshold cycle values vs. hematinconcentration for hematin inhibited samples using the 92 bp and the 172bp internal positive controls (IPC's), respectively.

FIGS. 6A and 6B show observed threshold cycle values vs. humic acidconcentration for humic acid inhibited samples using the 92 bp and the172 bp IPC's, respectively.

FIG. 7 shows the observed threshold cycle values vs. melaninconcentration for melanin inhibited samples using the 172 bp IPC.

FIG. 8 shows the effects of mechanical degradation by sonication asplots of relative fluorescence units across three different DNAconcentrations without degradation and for two degradation times.

FIG. 9 shows the effects of chemical degradation by the action of DNaseI as plots of relative fluorescence units across three different DNAconcentrations without degradation and for three degradation times.

FIG. 10 shows the effects of environmental degradation by the action ofambient heat and humidity for a period of five years as plots ofrelative fluorescence units across three different DNA concentrationsfor three degradation times.

FIG. 11 shows STR results for 1 ng targeted sonicated samples.

FIG. 12 shows STR results for 1 ng targeted DNase I treated samples.

FIG. 13 shows STR results for 200 pg targeted environmentally degradedsamples.

FIG. 14 shows species study results (striped red bars show long targetSVA; solid blue bars show short target Yb8).

FIG. 15 shows the amplification plot of the Y chromosome target sequencein a single-plex reaction.

FIG. 16A shows the amplification plot of the Y chromosome target in afour-target multiplex reaction.

FIG. 16B shows the standard curve of the Y chromosome target in afour-target multiplex reaction.

DETAILED DESCRIPTION OF THE INVENTION

One embodiment of the present invention is a process for quantitating ahuman DNA in a sample in order to assess the extent of degradation ofthe DNA therein, the process comprising the steps of providing a sampleto be analyzed, using a real time polymerase chain reaction (PCR) systemto separately quantitate within the sample a first retrotransposoninterspersed element and a second retrotransposon interspersed element,the first retrotransposon interspersed element being an Alu element, thesecond retrotransposon interspersed element being an SVA element of theRP gene, and calculating a ratio of an occurrence within the sample ofthe first retrotransposon interspersed element to an occurrence of thesecond retrotransposon interspersed element.

In another embodiment of the present invention, the secondretrotransposon interspersed element used as a target for quantitationwith a real time PCR system may comprise at least three times as manybase pairs as are comprised by the first retrotransposon interspersedelement.

In another embodiment of the present invention, the firstretrotransposon interspersed element may be a target sequence that hasabout 80 base pairs and is an Alu element of subfamily Yb8, and thesecond retrotransposon interspersed element may be a target sequencethat has about 290 base pairs and is an SVA element of the RP gene.

In another embodiment of the present invention, the inventive processesmay be used to determine the extent of admixture of male and female DNAin a test sample. This embodiment is enabled by the existence of a malespecific target sequence having about 90 base pairs, the male specifictarget sequence being deleted on the human X-chromosome in an X-Ychromosome homologous region and being therefore specific to male DNA.As explained in the disclosure of Walker, et al., U.S. Pat. No.7,405,044 (hereinafter ‘Walker’), which is hereby incorporated byreference in its entirety, sex chromosome assays may be designed arounda 90 bp deletion on the human X-chromosome in the X-Y homologous region(col. 5, ln 42-44). In FIG. 3 of Walker, the relevant primers are shownin bold font and the chromosome specific probes are shown in lower caseunderlined font. The deletion starts at X position 89810740 asdetermined by BLAT (The BLAST like Alignment Tool) (Walker, col. 5, ln44-47).

The process of an embodiment of the invention may further comprise thesteps of providing a first probe, the first probe comprising a firstmoiety capable of fluorescence at a first diagnostic wavelength and afirst quencher capable of quenching the first moiety fluorescence, thefirst probe being targeted to the first retrotransposon interspersedelement, providing a second probe, the second probe comprising a secondmoiety capable of fluorescence at a second diagnostic wavelength and asecond quencher capable of quenching the second moiety fluorescence, thesecond probe being targeted to the second retrotransposon interspersedelement, providing at least one primer that is useful in a real-timepolymerase chain reaction system, the system being capable ofamplification of a DNA sample, providing a Taq polymerase enzyme capableof catalyzing the formation of a nucleic acid sequence that iscomplimentary to one present in the sample, the Taq polymerase enzymebeing capable both of cleaving the first probe to separate the firstfluorescent moiety from the first quencher and of cleaving the secondprobe to separate the second fluorescent moiety from the secondquencher, treating the sample with the first probe and the second probe,amplifying the sample using the at least one primer and the Taqpolymerase enzyme by means of the real-time polymerase chain reactionsystem, illuminating the sample during each real time polymerase chainreaction cycle using an excitation source capable of inducingfluorescence in both the first moiety and the second moiety, measuringthe fluorescence emitted from the first moiety and the fluorescenceemitted from the second moiety for each real time polymerase chainreaction cycle, determining a threshold cycle number for the firstretrotransposon interspersed element and the second retrotransposoninterspersed element, and comparing the determined threshold cyclenumbers with standard curves (threshold cycle number vs. quantity ofDNA) for each of the first retrotransposon interspersed element and thesecond retrotransposon interspersed element to determine a concentrationfor each of the first retrotransposon interspersed element and thesecond retrotransposon interspersed element within the sample.

In another embodiment of the present invention, the goal of determiningthe extent of degradation present in a DNA sample may be realized byusing two independent genomic targets, obtaining quantification of ashort DNA fragment having about 79 base pairs and a long DNA fragmenthaving about 290 base pairs in a degraded DNA sample. A multi-copy intraAlu based approach has been developed using these target fragments toquantify human specific DNA in an evidence sample and has beensuccessfully used to obtain DNA quantification with high sensitivity.The use of internal primers to amplify DNA segments including that of anAlu element allows for human specificity as well as high sensitivitywhen compared to a single copy target.

The method for quantifying the extent of degradation of a human DNAsample relies on the fact that the integrity of the longer insertionsequences will be disrupted at a faster pace than will the integrity ofthe shorter insertion sequences as the DNA sample degrades in theenvironment. As the polymerization of the PCR reaction proceeds and thetwo TaqMan fluorescent probes are cleaved, the respective fluorescentsignals are monitored during each PCR cycle, and a threshold cycle, thecycle upon which the signal is first detectable, is determined for eachtarget. Using the log linear relationship between threshold cycle andDNA concentration, a concentration for each target sequence may bedetermined, and the concentration ratio of the respective targetsequences in the DNA sample may be determined. This ratio will be anindication of the extent of degradation of the sample.

In an embodiment of the invention, primers and TaqMan probes aredesigned using two independent insertion targets. The first is arelatively short (50-150 base pairs) retrotransposon interspersedelement insertion, whereas the second is a relatively longer (150-500base pairs) retrotransposon interspersed element insertion. In otherembodiments, the first insertion target has 60-125 base pairs, 75-85base pairs, or 79 base pairs, and the second insertion target has200-400 base pairs, 220-320 base pairs, 280-300 base pairs, or 290 basepairs.

The retrotransposon target elements of the present invention must beselected with care. Experimentation with a multiplex composed of Yb8 (80bp) and Ya5 (250 bp) targets exhibited cross reactivity due to thesequence similarities of these targets. Quantitation values of samplesamplified with individual targets Yb8 and Ya5 were discrepant whencompared to quantitation values of the same samples amplified in amultiplex reaction containing primers and probes for both the short(Yb8) and long (Ya5) targets in a single amplification due to this crossreactivity. For this study, two forward primers (SEQ ID NO: 1 and SEQ IDNO: 2), a reverse primer (SEQ ID NO: 3) and a probe (SEQ ID NO: 4)corresponding to the Ya5 250 base pair fragment were developed.

In a preferred embodiment of the invention, the shorter retrotransposoninterspersed element is a 79 base pair sequence from the Yb8 subfamilyof Alu insertions, and the longer retrotransposon interspersed elementis a 290 base pair sequence of an SVA element. In this embodiment, asystem was developed using the Yb8 Alu sequence of 79 bp in size for theshort fragment labeled in 5-carboxyfluorescein (FAM) and the SINE-Rregion of SVA sequence (H. Wang, et. al, J. Mol. Biol. 354: 994-1007(2005)) of 290 bp in size labeled in Cy5 for the long target. Asynthetic sequence labeled with indocarbocyanine Cy3 dye was also usedas an internal positive control (IPC) to assess the presence or absenceof inhibitors in the sample. A second version of this embodimentincludes a fourth, male DNA specific, target. In this system, the 79 bpYb8 fragment was labeled in2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE), the 290 bpSVA fragment was labeled in indocarbocyanine Cy5, the male DNA targetsequence on the Y chromosome is a 76 bp fragment and was labeled in FAM,and the IPC sequence was labeled in Cy3. In other embodiments making useof similar approaches, other multi-copy retrotransposons, such as LongInterspersed Elements (LINE), may also be used.

In some embodiments, use of a multi-copy target, two different sizesequence markers along with a synthetic target as an Internal PositiveControl (IPC) may provide an additional assessment for the presence ofPCR inhibitors in the test sample. This is an important way ofdetermining whether the sample matrix may be altering the operation ofthe test by providing assurance that the expected fluorescence ratiosare obtainable under the test conditions.

The present invention will now be described more fully with reference tothe accompanying drawing (FIG. 1), in which an exemplary embodiment ofthe invention is shown.

As shown in FIG. 1, a DNA sample (S100) is paired with three TaqManprobes (S110, S120, and S130), a real-time PCR primer (S140) and a Taqpolymerase enzyme (S150), the Taq polymerase enzyme being capable ofcleaving the TaqMan probes, separating each fluorescent moiety from itsrespective quencher. In the TaqMan procedure, the sample is treated withthe first TaqMan probe, the second TaqMan probe and the third TaqManprobe (S160), the TaqMan probes anneal with one of the complimentarynucleic acid strands (S170, S180, S190), the sample is amplified using astandard real-time PCR technique, and the TaqMan probes corresponding tothe target elements are cleaved to separate the three fluorophores fromtheir respective quenchers during repetitive denaturation and annealingsteps (S200). Illuminating the sample during each PCR cycle causes thefluorophores from both types of cleaved TaqMan probes to fluoresce. Athreshold cycle for each of the Yb8 element, the SVA element and theinternal positive control may then be determined, and concentrations ofeach of these may then be determined by comparison with standard curves(S210). The ratio of concentrations of the Yb8 element and the SVAelement may be related to the extent of environmental degradation in theoriginal DNA sample (S220). Comparison of the obtained concentration ofthe SVA element with a concentration obtained for the internal positivecontrol provides information about the extent to which PCR inhibitorsmay be present in the sample.

The latter process is versatile and may be modified to provideadditional useful information. One of skill in the art may readilyenvision modification of the above procedure to include a fourth probecorresponding to the male specific DNA target described above.

Precision and sensitivity studies have indicated that the process of thepreferred embodiment of the present invention has a sensitivitythreshold in the range of about 5 pg to about 9 pg, similar tosensitivities reported for other Alu-based quantification systems. Theamount of synthetic IPC target may be adjusted to provide reproduciblethreshold cycle (Ct) values in the range from 18 to 22 cycles forsamples with no inhibition.

Estimated quantitation for both 79 bp and 290 bp fragments obtainedusing the new primers and TaqMan probes were compared with STR analysisresults obtained from DNA samples degraded using sonication, DNAse I andenvironmental degradation, and the correlation indicates that theprocess of the preferred embodiment of the present invention is areliable one for determining the extent of degradation of a human DNAsample. In all instances, the STR results mirrored the degradationratios calculated by this duel target quantification assay.

The present invention provides a quantification system that accuratelyassesses the quality of human DNA present in a forensic sample. Resultsdemonstrate the preferred embodiment of the present invention to bespecific to higher primates, sensitive down to 5-9 pg of DNA,reproducible, and a useful tool for assessing degradation in abiological sample. A DNA based qualitative/quantitative/inhibitionassessment system that accurately predicts the status of a biologicalsample may serve as a valuable tool for deciding which DNA test kit toutilize when processing forensically compromised samples for DNAtesting.

EXAMPLES

Preferred embodiments of the present invention may be better understoodthrough the following characterizations of experimental methods,conditions, accuracy and precision of the process of the presentinvention and the associated examples that follow.

Example 1 Primers and Probes

Primers and probes that are useful in the described embodiments areshown in Table 1. An intra RE primer design was used to target a Yb8 Alusequence of 79 bp in size for the “short” fragment as well as a sequencein the SINE-R region of SVA of 290 bp in size for the “long” target. Aninternal positive control (IPC) to assess the presence or absence ofinhibitors in the sample was studied as well. IPC target syntheticsequences of 92 bp, 158 bp, 172 bp, and 192 bp were studied. Inhibitionstudies using inhibitors commonly found in forensic samples wereperformed on the 92 bp, 158 bp, and 172 bp IPC target sequences.

Two systems were developed: one three target system containing the Yb8“short” Alu fragment labeled in FAM, the SVA “long” fragment labeled inCy5, and a synthetic sequence labeled with Cy3 dye used as the IPC. Asecond system comprised of four targets was developed incorporating amale specific DNA target sequence to detect male DNA in the sample. Inthis system, the “short” 79 bp Yb8 fragment was labeled in JOE, the“long” 290 bp SVA fragment was labeled in Cy5, the male specific DNAtarget sequence on the Y chromosome is a 76 bp fragment and was labeledin FAM, and the IPC sequence was labeled in Cy3.

Primers and probes were designed for the Y chromosome marker to providespecificity to the Y chromosome target by placing the probe within the90 bp X chromosome deletion. Single-plex reactions were run to verifythe correct products formed. Because the copy number of this targetsequence is not as high as the Yb8 or SVA copy number, a higher numberof cycles were needed to produce amplification products. In a singlereaction, 40 cycles produced a standard curve with 126% efficiency. SeeFIG. 15 for the real-time amplification plot of the single-plex Yreactions.

TABLE 1 Primer sequences Primer or Probe Target Name NamePrimer Sequence Ya5 Forward-1 TCACGCCTGTAATCCCAGCACTT Forward-2ACGCCTGTAATCCCAGCACTTTG Reverse TCTGTCGCCCAGGCTGGAGT ProbeATCACGAGGTCAGGAGATCGAGACCAT Yb8 Forward GGAAGCGGAGCTTGCAGTGA ReverseAGACGGAGTCTCGCTCTGTCGC Probe AGATTGCGCCACTGCAGTCCGCAG SVA ForwardTGGGATCCTGTTGATCTGTGACCT Reverse GATTTGGCAGGGTCATGGGACAAT ProbeAAGGGCGGTGCAAGATGTGCTTTGTT Forward-Q2F ATGTGCTGTGTCCACTCAGGGTTAReverse-Q2R1 TTCTTGGGTGTTTCTCACAGAGGG Reverse-Q2R2ATTCTTGGGTGTTTCTCACAGAGG Forward-Q3F CCAACCCTGTGCTCTCTGAAAC Reverse-Q3RTTTGGCAGGGTCATGGGACAA IPC Forward (90) AAAGATCCTGCCAACAGGACAGTGReverse (90) ACAGACGGTATAGAGACCAATCAG IPCr7-Forward (158)GCATAAAGATCCTGCCAACAG IPCr7-Reverse (158) ACCAAAGTGCTGCAGAAATAC ProbeAGGCAGAGATTGCACTGCCTTAAAGTGG IPC-M Forward GCATAAAGATCCTGCCAACAGReverse - 172 bp GCCCGAACTTCCAACACTAT Reverse - 192 bpATTGTTCCTCCTGCCTGATT Probe ACAGTGTCAGGCAGAGATTGCACT Y chromosome ForwardCAATGTG[CTAGGCTCTAGGAATAC Reverse AAGAGTGTCATGGCTCAAAGAG ProbeAGAGAGTATGACAAACATGGCATGGGC

Optimization of PCR conditions was carried out as described in Examples2-5.

Example 2 Number of Cycles

The manufacturer recommendation for the QPCR Multiplex master mix is:95° C. for 10 minutes followed by 40 cycles of 95° C. for 15 seconds and60° C. for 60 seconds. To test the number of cycles that would beoptimal for the duel target quantification assay, the number of cycleswas varied. Tests of 40 cycles and 32 cycles were carried out, and PCRefficiency values were examined. The results showed higher PCRefficiencies when 32 cycles were used.

Example 3 Annealing and Denaturation Times

The manufacturer recommendation for PCR conditions is: 95° C. for 10minutes followed by 40 cycles of 95° C. for 15 seconds and 60° C. for 60seconds. To test which PCR conditions would be optimal for the InnoQuantQuantification Kit (InnoGenomics Technologies, LLC), denaturation andannealing times were varied. Four annealing times were tested: 45, 60,70, and 120 seconds; and two denaturation times were tested: 15 secondsand 30 seconds. PCR efficiency, R² values, and standard deviation of thetriplicate quantitation values (three DNA extracts were run intriplicate) were examined.

Results indicate that, for the preferred embodiment of the presentinvention, the longer second annealing time yields slightly improved PCRefficiencies and lower standard deviation of the triplicatequantification values than was achieved at shorter annealing times. Alonger annealing time also provides more time for the enzyme to functionproperly with the longer targets. Additionally, results indicate that a30 second denaturation time significantly decreases the R² value of thestandard curve of both targets relative to that attained with the 15second denaturation time. Therefore, the 15 second denaturation time and120 second annealing time were selected for use with the preferredembodiment.

Example 4 Annealing Temperature

Three annealing temperatures were tested: the manufacturer-recommended60° C., 61° C., and 62° C. Efficiencies, R² values, and standarddeviation of the sample triplicates were analyzed. The results are shownin Table 2.

TABLE 2 Annealing temperature runs Yb8 Yb8 Yb8 SVA SVA SVA Run dateConditions Efficiency R² SD Efficiency R² SD Oct. 30, 2012 SVA/Yb8 0.3IPC 88% 0.998 0.29 87% 0.998 0.39 0.2 stds at 20 ng 60° C. Annealingtemp Oct. 30, 2012 SVA/Yb8 0.3 IPC 95% 0.993 0.16 85% 0.994 0.24 0.2stds at 20 ng 61° C. Annealing temp Oct. 31, 2012 SVA/Yb8 0.3 IPC 91%0.998 0.27 82% 0.997 0.22 0.2 stds at 20 ng 61° C. Annealing temp Oct.31, 2012 SVA/Yb8 0.3 IPC 86% 0.996 1.51 84% 0.997 0.98 0.2 stds at 20 ng62° C. Annealing temp

A 62° C. annealing temperature exhibited wide standard deviation of thesample triplicates and a relatively low efficiency of the Yb8 target.Based on these results, an annealing temperature of 61° C. was selecteddue to the relatively high efficiency of the Yb8 target. The final PCRconditions selected for the preferred embodiment were as follows: 95° C.for 10 min, 32 cycles of: 95° C. for 15 seconds, 61° C. for 70 seconds.

Example 5 Primer and Probe Concentrations

The two targets were tested individually to determine the best primerand probe concentrations. Once a determination was made from theindividual runs, the two targets were multiplexed, and varyingconcentrations of each in combination were tested. At least two separateruns were performed for each parameter to ensure reproducibility of theresults. Primer and probe concentrations were varied for the SVA and Yb8targets. (The IPC primer/probe concentrations remained fixed at 150 nMfinal concentration in the reaction [0.2 μL put into reaction], and IPCtemplate DNA amount remained fixed at 4.5 pg.) The average of thestandard curve efficiencies and R² values, and standard deviation of thesample quantities (tested in triplicate) were examined. Based on theresults, a final primer concentration of 0.3 μM (0.6 μl of a 10 μMstock) for SVA and 0.25 μM (0.5 μl of a 10 μM stock) for Yb8 in a totalreaction volume of 20 μl was selected.

Example 6 Primer Cross Reactivity

To verify the accuracy of the quantity obtained from each target and torule out any cross reactivity between primers, a melt curve analysis andamplification artifacts from reactions containing the short targetforward primer and the long target reverse primer were examined byfragment analysis. Melt curves exhibit the presence of a single peak foreach of the Yb8 and SVA reactions, as expected. A slightly broad basewas observed for both targets, indicating the presence of primer dimers.FIGS. 2A and 2B show melt curve analyses for the Yb8 and SVA targetsrespectively.

Fragment analysis confirmed the presence of the targeted fragment foreach target as well as the absence of any artifacts resulting from crossreactivity between the primers (see Table 3). Some primer dimers wereobserved and one artifact at ˜67 bp was also observed. Overall, theresults of the melt curve analysis and the fragment analysis confirm thepresence of the targeted fragments and the absence of artifacts.

TABLE 3 Fragment Analysis Results Peaks Observed (size in base pairs)Primer Dimer Expected Peak Artifact SVAF/R + 50 bp & 73 + 290 Yb8F/R59/65 bp SVAF/R +  ~50 bp 290 67 Yb8R Yb8F/R + 59/65 bp  73 SVAR SVAF +Yb8R No peaks observed 67 Yb8F + SVAR No peaks observed

Example 7 Multiplex Sensitivity

The sensitivity study was designed such that a given set of measurements(threshold cycle, or C_(t) value) could be evaluated at various inputranges using a log-linear relationship across the input amounts and toestablish the lowest levels of sensitivity where the log-linearrelationship was lost. For the Sensitivity Study, one plate of samples,standards, and negative controls, that were quantified with theInnoQuant Quantification Kit and run on an Applied Biosystems 7500Real-Time PCR System, were evaluated using the HID Real-Time PCRAnalysis Software v 1.1. Three serial dilutions of the standards(designated as STD1, STD2, and STD3) were made using Teknova DNAdilution buffer (10 mM Tris-0.1 mM EDTA) and run on the trays induplicate. Different combinations of the DNA standards from the 2quantification plates were used to determine the average C_(T) andquantity of each standard when designated as “unknown”. An extension ofthe DNA standard dilution STD3 was made two quantities above the higheststandard and two quantities below the lowest standard at 100 ng/μl, 50ng/μl, 0.0045 ng/μl and 0.0023 ng/μl concentrations. Using the differentDNA standard configurations, an average C_(T) and quantity werecalculated for each standard labeled as an unknown. The C_(T) vs. DNAquantity (ng/μl, on a logarithmic scale) for the unknown standardsamples were plotted to demonstrate the linearity of the system. Thesame analysis was performed on other samples run during this validation,including the 100 ng/μl, 50 ng/μl, 0.0045 ng/μl and 0.0023 ng/μlextensions of the standard samples.

Example 8 Accuracy, Precision, Sensitivity and Linearity of DNADeterminations

Table 4 shows the average quantity and C_(T) values determined for thestandards dilutions using the different combinations of the standardcurves run on the plate. FIG. 3 is the graphical representation of thedata in Table 4. Results demonstrate that the InnoQuant Kit consistentlydetected all samples at as little as 2.3 pg of total input DNA. As theDNA concentration goes beyond the concentrations of the standard curve(20 ng/ul to 0.009 ng/ul), the variation from the expected quantitiesincreases, and linearity is lost to an extent. However, these variationsare minimal and would not have affected the ability to obtain full DNAprofiles; the increase variation can be expected with the lowerconcentrations (stochastic amplification effects). The average foldchange between the quantity and the expected quantity is 1.13 for theshort target and 1.07 for the long target, when all the dilutions areincluded. A 1.13 fold change of the human average quantity value wouldresult in an approximate 13% variation in the quantity of the humanquantifications. The two targets are amplified in a single reaction, butthe small target quantity demonstrated slightly higher variation atseveral of the sensitivity dilutions. When examining the samples withinthe concentrations of the standard curve (20 ng/ul to 0.009 ng/ul), thefold changes decrease to an average of 1.04 for the short target and1.06 for the long target. These fold changes in quantities would notsignificantly affect the Relative Fluorescent Units (RFU) seen in theelectropherograms and are due to stochastic amplification effects.Higher fold differences were observed outside of the standard curverange and would be expected with the amount of template amplified.

The results for the 0.0045 ng and 0.0023 ng sample demonstrate thesensitivity of this dual target quantification assay to detect DNAsamples below 9 pg. The reproducibility of such values is influenced bystochastic amplification effects associated with low templateamplifications. The stochastic amplification effects can be seen inhuman or male amplifications and not necessarily at the same time.Samples at or near the ends of the standard curve are more susceptibleto changes in slope of the standard curve. When samples quantify greaterthan 20 ng, the laboratory may consider diluting and re-quantifyingsamples to assure appropriate quantification value.

TABLE 4 Average quantity and Ct values for the standard dilutions andthe gDNA dilutions Sample Name (Expected Concen- Observed Yb8 ObservedSVA tration) Ave. Ave. Fold Ave. Ave. Fold ng/ul CT Qty Change CT QtyChange 100 9.41 153.8067 1.54 13.73 104.7562 1.05 50 10.43 78.2061 1.5614.78 50.5777 1.01 20 12.57 18.7944 1.06 16.00 22.4396 1.12 6.7 14.086.7444 1.01 17.96 6.1197 1.09 2.2 15.66 2.3506 1.07 19.54 2.1496 1.020.74 17.33 0.7682 1.04 21.13 0.7456 1.01 0.25 18.93 0.2625 1.05 22.900.2318 1.08 0.08 20.64 0.0836 1.05 24.38 0.0866 1.08 0.027 22.34 0.02681.01 26.09 0.0278 1.03 0.009 24.00 0.0088 1.02 27.78 0.0091 1.02 0.004525.09 0.0043 1.06 28.61 0.0052 1.16 0.0023 29.66 0.0021 1.09 29.660.0026 1.13 Average Yb8 Fold 1.13 Average SVA Fold 1.07 Change Change

Example 9 Concordance and Reproducibility

Nineteen samples were quantified using this dual target assay, thepreferred embodiment of the invention, and the Quantifiler® Human kitfrom Life Technologies. Quantifiler® human DNA concentrations averaged140% of those calculated using the short target (Yb8) of this dualtarget assay. If differences were observed, in all instances,Quantifiler® human values were higher than dual target quantificationassay values. These differences are attributed to differences in the DNAstandards and differences in amplicon length (62 bp in Quantifiler® vs.80 bp in dual target quantification assay system).

To test the reproducibility of the system across various DNAconcentrations, the sensitivity data was examined for fold changes inconcentration between the replicates. FIG. 4 shows the results of thefold changes between each replicate of the standard dilutions (at least34 readings from each concentration). Results indicate that between theconcentrations of the standards (20 ng to 9 pg), fold changes are allless than 1.20, which is equivalent to a 20% variation. As theconcentrations go above or below the optimal range, fold changesincrease, as expected.

Example 10 Effects of Inhibition

Varying concentrations of humic acid and hematin were used to inhibitsamples using the 92 base pair IPC sequence and the 172 bp IPC sequence.Varying concentrations of melanin were used to inhibit samples using the172 bp IPC sequence. This inhibitor was selected due to the prevalenceof hair samples in forensic casework. The samples were quantified withthe InnoQuant Quantification Kit™, and the C_(t) values of the Yb8, SVA,and IPC targets were evaluated. FIGS. 5A, 5B, 6A, 6B and 7 show theresults for the inhibitors tested.

A comparison of the C_(t) values between the two sized IPC targets forthe same inhibitor concentrations shows the 172 bp target is inhibitedmore readily, as expected due to its larger size. Results show a gradualincrease in C_(t) as the concentration of the inhibitor increases withboth the 92 and the 172 bp targets. With the 92 bp IPC, it is observedthat the Yb8 and IPC targets have similar reactions to inhibitors, andare affected at approximately the same level of inhibitor. SVA has ahigher sensitivity to inhibitors and is the first target to be affected.In contrast, the 172 bp IPC target sequence is the first target of thethree to be affected by inhibition. The 172 bp IPC target tracks withthe SVA target, as both are affected similarly as the concentration ofthe inhibitor increases, whereas Yb8 is the last target to be affectedas the concentration of the inhibitor increases. Due to the improvedcorrelation of the 172 bp IPC with the short and long targets, this wasselected to be incorporated into the multiplex.

As with the other inhibitors tested, results of the melanin inhibitorshow a gradual increase in the Ct of all three targets as theconcentration of the inhibitor increases. In the case of melanin, theSVA long target is the first of the three targets to be affected,possibly due to a different mechanism of inhibition.

Example 11 Effects of Degradation on the Determination of DNA

Degradation studies were performed to assess the utility of this dueltarget quantification assay, the preferred embodiment of the presentinvention, with degraded samples. The experiments were designed toassess three types of degradation: mechanical (via sonication), chemical(via DNase I) and environmental degradation (samples placed in theoutside elements [heat, humidity] for a period of 5 years). Degradationratios based on the observed quantities of the long and short targetswere expressed as a percentage=(1−[Long Qty/Short Qty])*100. Results areshown in FIGS. 8 through 10.

Downstream STR analysis of the degraded samples was performed using theIdentifiler® Plus STR kit (Applied Biosystems/Life Technologies),targeting 3 different DNA concentrations: 1 ng (manufacturer recommendedtarget amount), 500 pg, and 200 pg. In all instances, the STR resultsmirrored the degradation ratios calculated by this duel targetquantification assay, the preferred embodiment of the present invention.As the degradation increased, the typical “ski slope” effect wasobserved in the STR results, until eventually, no results (or verypartial, inconclusive results) were observed at the highest extent ofthe degradation. The extent of the degradation was observed to a muchhigher level at the low concentrations of DNA (500 pg and 200 pg). Asexpected, lower RFUs and in some instances, no results, were observedwhen the DNA was both degraded and low in amount. FIGS. 11 through 13show the STR results of the degradation study.

Example 12 Species Specificity

A quantification system used for forensic DNA samples must not react tonon-primate DNA, as the STR systems commonly used in crime laboratoriesare only reactive to human and primate DNA. Three primates, sevennon-primate mammals, and five prokaryotic species were analyzed usingthe InnoQuant Quantification Kit™, the analyses including Yb8, SVA andthe 172 bp IPC target. DNA purified from two species of dogs, two cats,deer, rat, mouse, mosquito, chicken, green monkey, chimpanzee,orangutan, Escherichia coli, Ralstonia eutropha, Rhodococcus rubber,yeast, and Staphylococcus aureus were run in duplicate. The DNA sampleswere at 5 ng/μl. A sample was considered “reactive” if >1 pg/μl of DNAwas detected with the process of the present invention using a human DNAstandard curve.

Of the species tested, only higher primate samples were reactive (FIG.14). Cross reactivity of non-human primate species with the commonlyused STR systems has been previously demonstrated. These resultsdemonstrate that the process of the present invention is adequatelyspecies-specific for forensic use and does not yield quantitativeresults with non-primate samples.

Example 13 Four Target System (Yb8, SVA, Y Deletion, and IPC)

The addition of a male specific target to the real-time PCR quantitationmultiplex is useful to detect the amount of male DNA in samples thatcontain large quantities of female DNA and low quantities of male DNA. Aregion of the human Y chromosome DNA containing a 90 base pair sequencewhich is deleted on the human X-chromosome in an X-Y chromosomehomologous region was selected by using a primer pair specific to thehuman Y chromosome DNA in order to obtain amplified products (Walker, etal., Anal. Biochem. 337(1): 89-97 (2005)).

Example 14 Multiplex Reaction

When added to the multiplex, the male specific fragment is labeled inFAM, the “short” Yb8 fragment in JOE, the “long” SVA fragment in Cy5 andthe 158 bp internal positive control (IPC) to assess the presence orabsence of inhibitors in the sample in Cy3. The cycle number of themultiplex was increased from 32 to 35 and 38. Thirty-eight cyclesproduced adequate results, with efficiencies of the three targets, SVA,Yb8, and Y, at 88%, 99%, and 94%, respectively. See FIGS. 16A and 16Bfor the amplification plot and standard curve, respectively, of the Ychromosome target in a four-target multiplex reaction.

While the invention has been described in connection with specific andpreferred embodiments thereof, it is capable of further modificationswithout departing from the spirit and scope of the invention. Thisapplication is intended to cover all variations, uses, or adaptations ofthe invention, following, in general, the principles of the inventionand including such departures from the present disclosure as come withinknown or customary practice within the art to which the inventionpertains, or as are obvious to persons skilled in the art, at the timethe departure is made. It should be appreciated that the scope of thisinvention is not limited to the detailed description of the inventionhereinabove, which is intended merely to be illustrative, but rathercomprehends the subject matter defined by the following claims.

What is claimed is:
 1. A process for quantitating human DNA in a sample in order to assess the extent of degradation of the DNA therein, the process comprising the steps of: providing a sample to be analyzed; using a real time polymerase chain reaction system to separately quantitate within the sample a first retrotransposon interspersed element and a second retrotransposon interspersed element, the first retrotransposon interspersed element being an Alu element, the second retrotransposon interspersed element being an SVA element of the RP gene; and calculating a ratio of an occurrence within the sample of the first retrotransposon interspersed element to an occurrence of the second retrotransposon interspersed element.
 2. The process according to claim 1, the quantitation of the first retrotransposon interspersed element and the second retrotransposon interspersed element being performed simultaneously.
 3. The process according to claim 2, further comprising the step of using the ratio to determine an extent of degradation of the DNA in the sample.
 4. The process of claim 1, the second retrotransposon interspersed element comprising at least three times as many base pairs as are comprised by the first retrotransposon interspersed element.
 5. The process of claim 2, further comprising the steps of: providing a first probe, the first probe comprising a first moiety capable of fluorescence at a first diagnostic wavelength and a first quencher capable of quenching the first moiety fluorescence, the first probe being targeted to the first retrotransposon interspersed element; providing a second probe, the second probe comprising a second moiety capable of fluorescence at a second diagnostic wavelength and a second quencher capable of quenching the second moiety fluorescence, the second probe being targeted to the second retrotransposon interspersed element; providing at least one primer that is useful in the real-time polymerase chain reaction system, the system being capable of amplification of a DNA sample; providing a Taq polymerase enzyme capable of catalyzing the formation of a nucleic acid sequence that is complimentary to one present in the sample, the polymerase enzyme being capable both of cleaving the first probe to separate the first fluorescent moiety from the first quencher and of cleaving the second probe to separate the second fluorescent moiety from the second quencher; treating the sample with the first probe and the second probe; amplifying the sample using the at least one primer and the Taq polymerase enzyme by means of the real-time polymerase chain reaction system, the real-time polymerase chain reaction system including a plurality of polymerase chain reaction cycles; illuminating the sample during each real-time polymerase chain reaction cycle using an excitation source capable of inducing fluorescence in both the first moiety and the second moiety; measuring the fluorescence emitted from the first moiety and the fluorescence emitted from the second moiety for each real time polymerase chain reaction cycle; determining a threshold cycle number for the first retrotransposon element and the second retrotransposon element; and comparing the determined threshold cycle numbers with standard curves for each of the first retrotransposon element and the second retrotransposon element to determine a concentration for each of the first retrotransposon element and the second retrotransposon element within the sample.
 6. The process of claim 5, the step of providing at least one primer comprising: providing at least one primer selected from the group consisting of a forward primer labeled SEQ ID NO: 5 and a reverse primer labeled SEQ ID NO: 6; and providing at least one primer selected from the group consisting of a forward primer labeled SEQ ID NO: 8, a forward primer labeled SEQ ID NO: 11, a forward primer labeled SEQ ID NO: 14, a reverse primer labeled SEQ ID NO: 9, a reverse primer labeled SEQ ID NO: 12, a reverse primer labeled SEQ ID NO: 13 and a reverse primer labeled SEQ ID NO: 15: (SEQ ID NO: 5) 5′ GGAAGCGGAGCTTGCAGTGA 3′ (SEQ ID NO: 6) 5′ AGACGGAGTCTCGCTCTGTCGC 3′ (SEQ ID NO: 8) 5′ TGGGATCCTGTTGATCTGTGACCT 3′ (SEQ ID NO: 9) 5′ GATTTGGCAGGGTCATGGGACAAT 3′ (SEQ ID NO: 11) 5′ ATGTGCTGTGTCCACTCAGGGTTA 3′ (SEQ ID NO: 12) 5′ TTCTTGGGTGTTTCTCACAGAGGG 3′ (SEQ ID NO: 13) 5′ ATTCTTGGGTGTTTCTCACAGAGG 3′ (SEQ ID NO: 14) 5′ CCAACCCTGTGCTCTCTGAAAC 3′ (SEQ ID NO: 15) 5′ TTTGGCAGGGTCATGGGACAA 3′.


7. The process of claim 5, the first probe having a sequence labeled SEQ ID NO: 7 and the second probe being selected from the group consisting of a sequence labeled SEQ ID NO: 4 and a sequence labeled SEQ ID NO: 10: (SEQ ID NO: 4) 5′ ATCACGAGGTCAGGAGATCGAGACCAT 3′ (SEQ ID NO: 7) 5′ AGATTGCGCCACTGCAGTCCGCAG 3′ (SEQ ID NO: 10) 5′ AAGGGCGGTGCAAGATGTGCTTTGTT 3′.


8. The process of claim 5, the real time polymerase chain reaction system operating under the following approximate conditions: 95° C. for 10 minutes; 32-40 cycles of: 95° C. for 15 seconds, 61° C. for 70-120 seconds.
 9. The process of claim 5, further comprising the steps of: adding an internal positive control to the sample to form a sample mixture; using the real time polymerase chain reaction system to quantitate the internal positive control within the sample mixture; comparing an occurrence within the sample mixture of the second retrotransposon interspersed element with an occurrence within the sample mixture of the internal positive control; and using the comparison to determine an extent to which the process was affected by the presence of an inhibitor in the sample.
 10. The process according to claim 9, the quantitation of the first retrotransposon interspersed element, the second retrotransposon interspersed element and the internal positive control being performed simultaneously.
 11. The process of claim 9, the internal positive control comprising a synthetic nucleotide sequence.
 12. The process of claim 9, the step of providing at least one primer comprising providing a primer for the internal positive control, the primer for the internal positive control being selected from the group consisting of a sequence labeled SEQ ID NO: 16, a sequence labeled SEQ ID NO: 17, a sequence labeled SEQ ID NO: 18 and a sequence labeled SEQ ID NO: 19: (SEQ ID NO: 16) 5′ AAAGATCCTGCCAACAGGACAGTG 3′ (SEQ ID NO: 17) 5′ ACAGACGGTATAGAGACCAATCAG 3′ (SEQ ID NO: 18) 5′ GCATAAAGATCCTGCCAACAG 3′ (SEQ ID NO: 19) 5′ ACCAAAGTGCTGCAGAAATAC 3′.


13. The process of claim 9, further comprising providing a third probe, the third probe comprising a third moiety capable of fluorescence at a third diagnostic wavelength and a third quencher capable of quenching the third moiety fluorescence, the third probe being targeted to the positive internal control, the Taq polymerase enzyme being further capable of cleaving the third probe to separate the third fluorescent moiety from the third quencher, the third probe having a sequence labeled SEQ ID NO: 20: (SEQ ID NO: 20) 5′ AGGCAGAGATTGCACTGCCTTAAAGTGG 3′ .


14. The process of claim 1, the first retrotransposon interspersed element being one of a SINE target sequence, a LINE target sequence and a SVA target sequence, the first retrotransposon interspersed element consisting of 40 to 150 base pairs, the second retrotransposon interspersed element being one of a SINE target sequence, a LINE target sequence and a SVA target sequence consisting of 120 to 400 base pairs.
 15. The process of claim 1, the sensitivity of the DNA quantitation being in the range of from about 5 pg to about 9 pg.
 16. The process of claim 1, the efficiency of the real time polymerase chain reaction system with respect to each of the first retrotransposon interspersed element and the second retrotransposon interspersed element being at least about 80%.
 17. The process of claim 1, the step of using a real time polymerase chain reaction system including the preparation of standard curves for the quantitation of the first retrotransposon interspersed element and the quantitation of the second retrotransposon interspersed element, the standard curves each being a plot of a threshold cycle vs. a quantity of DNA on a logarithmic scale and each having an R² value of at least 0.99.
 18. The process of claim 1, the real time polymerase chain reaction system being substantially unreactive to non-primate DNA in the sample.
 19. The process of claim 1, the DNA having been degraded by mechanical means.
 20. The process of claim 1, the DNA having been degraded by chemical means.
 21. The process of claim 1, the DNA having been degraded by environmental means.
 22. The process of claim 1, the real time polymerase chain reaction system being one of SYBR® Green, TaqMan®, and AmpliFluor®.
 23. A process for quantitating total human DNA and male specific DNA in a sample in order to assess the extent of degradation of the DNA therein, the process comprising the steps of: providing a sample to be analyzed; using a real time polymerase chain reaction system to separately quantitate within the sample a male specific DNA sequence, a first retrotransposon interspersed element and a second retrotransposon interspersed element, the male specific DNA sequence being a 90 bp Y-chromosome specific DNA sequence, the first retrotransposon interspersed element being an Alu element, the second retrotransposon interspersed element being an SVA element of the RP gene; and calculating a ratio of an occurrence within the sample of the first retrotransposon interspersed element to an occurrence of the second retrotransposon interspersed element.
 24. The process of claim 23, the first retrotransposon interspersed element being one of a SINE target sequence, a LINE target sequence and a SVA target sequence consisting of 40 to 150 base pairs, the second retrotransposon interspersed element being one of a SINE target sequence, a LINE target sequence and a SVA target sequence consisting of 120 to 400 base pairs, and the male target being a region of a human Y chromosome DNA containing a 90 base pair sequence which is deleted on a human X-chromosome in an X-Y chromosome homologous region.
 25. The process of claim 23, the first retrotransposon interspersed element being a target sequence that has about 80 base pairs and is an Alu element of subfamily Yb8, the second retrotransposon interspersed element being a target sequence that has about 290 base pairs and is an SVA element of the RP gene, and the male specific DNA sequence being a region of a human Y chromosome DNA containing a 90 base pair sequence which is deleted on a human X-chromosome in an X-Y chromosome homologous region.
 26. The process of claim 25, the using step further comprising: providing at least one primer selected from the group consisting of a forward primer labeled SEQ ID NO: 5 and a reverse primer labeled SEQ ID NO: 6; providing at least one primer selected from the group consisting of a forward primer labeled SEQ ID NO: 8, a forward primer labeled SEQ ID NO: 11, a forward primer labeled SEQ ID NO: 14, a reverse primer labeled SEQ ID NO: 9, a reverse primer labeled SEQ ID NO: 12, a reverse primer labeled SEQ ID NO: 13 and a reverse primer labeled SEQ ID NO: 15: (SEQ ID NO: 5) 5′ GGAAGCGGAGCTTGCAGTGA 3′ (SEQ ID NO: 6) 5′ AGACGGAGTCTCGCTCTGTCGC 3′ (SEQ ID NO: 8) 5′ TGGGATCCTGTTGATCTGTGACCT 3′ (SEQ ID NO: 9) 5′ GATTTGGCAGGGTCATGGGACAAT 3′ (SEQ ID NO: 11) 5′ ATGTGCTGTGTCCACTCAGGGTTA 3′ (SEQ ID NO: 12) 5′ TTCTTGGGTGTTTCTCACAGAGGG 3′ (SEQ ID NO: 13) 5′ ATTCTTGGGTGTTTCTCACAGAGG 3′ (SEQ ID NO: 14) 5′ CCAACCCTGTGCTCTCTGAAAC 3′ (SEQ ID NO: 15) 5′ TTTGGCAGGGTCATGGGACAA 3′;

and providing at least one primer selected from the group consisting of a forward primer labeled SEQ ID NO: 25 and a reverse primer labeled SEQ ID NO: 26: (SEQ ID NO: 25) 5′ CAATGTG[CTAGGCTCTAGGAATAC 3′ (SEQ ID NO: 26) 5′ AAGAGTGTCATGGCTCAAAGAG 3′.


27. The process of claim 25, the using step further comprising providing a probe for the male specific DNA target sequence, the probe having a sequence labeled SEQ ID NO: 27: (SEQ ID NO: 27) 5′ AGAGAGTATGACAAACATGGCATGGGC 3′.


28. The process of claim 25, further comprising the steps of: adding an internal positive control to the sample to form a sample mixture; using the real time polymerase chain reaction system to quantitate the internal positive control within the sample mixture; comparing an occurrence within the sample mixture of the second retrotransposon interspersed element with an occurrence within the sample mixture of the internal positive control; and using the comparison to determine an extent to which the process was affected by the presence of an inhibitor in the sample.
 29. The process of claim 28, the step of using the real time polymerase chain reaction system to quantitate the internal positive control further comprising: providing at least one primer selected from the group consisting of a forward primer labeled SEQ ID NO: 21, a reverse primer labeled SEQ ID NO: 22, and a reverse primer labeled SEQ ID NO: 23: (SEQ ID NO: 21) 5′ GCATAAAGATCCTGCCAACAG 3′ (SEQ ID NO: 22) 5′ GCCCGAACTTCCAACACTAT 3′ (SEQ ID NO: 23) 5′ ATTGTTCCTCCTGCCTGATT 3′.


30. The process of claim 28, the step of using a real time polymerase chain reaction system to quantitate the internal positive control further comprising providing a probe for the positive internal control, the probe having a sequence labeled SEQ ID NO: 24: (SEQ ID NO: 24) 5′ ACAGTGTCAGGCAGAGATTGCACT 3′. 